This invention relates to a separator, and in particular to a cell separator for separating various cells on the basis of size.
There are several popular methods of separating or sorting cells, including (a) velocity sedimentation and/or density gradient centrifugation, (b) counter current distribution, and (c) selective agglutination.
Velocity sedimentation and density gradient centrifugation both rely upon the density (rather than size differences) of the cells or particles being separated. The density of the cells dictate the rates at which the cells drift through a substance of known density and viscosity during centrifuging. It has been found that the use of this method results in a significant variation in the sizes of the cells or particles isolated in any single collected fraction.
Counter current distribution relies upon lipid related membrance differences between cells. Such differences cause the cells to be more attracted to a positively or negatively charged medium. The method involves the use of two liquids, namely dextran solution and a polyethylene glycol solution. At present, there is no complete understanding of the biological significance or implication of separating cells on the basis of electrical charge. After sorting, the cells must be subjected to treatment with antibiotics. In certain cases, cell contact with antibiotics is undesirable.
In selective agglutination, ligens are bound to specific cell surface sugars. Cells sorted using this mehtod are either precipitated from solution or are attached to a ligen bound to a solid support. Only a few types of cells can be sorted using this method. Contact with the ligen must be as brief as possible and, under certain conditions, a ligen may behave as a mitogen which is completely undesirable. The continuous purches of ligen, sugars, equipment and operating medium is costly.
Another method of cell sorting involves the use of a multi-sectioned plastic sleeve defined by a plurality of plastic cylinders with screens and O-rings therebetween, and a retaining plate at the top end of the cylinders. The plate is secured with two wing nuts on long screws situated on each side of the cylinder. Separation medium is loaded into the sleeve from the top and flows through the screens for discharge from the bottom of the unit. The cells are loaded into the top of the sleeve and permitted to settle through screens of successively decreasing size. In order to harvest the cells, the unit must be dismantled from the top down. This method of separation is unsatisfactory for several reasons. During cell separation, the undisrupted flow of liquid is critical. During loading of the column, it is essential that no air remain trapped under any of the screens. Trapped air causes turbulence as liquids flow around these pockets, resulting in an artifactual separation. The particular O-ring plastic sleeve design often permits slight buckling to form in the surface of the screens which trap large bubbles of air. The sleeve is designed to be loaded from the top which often produces sealed chambers when mesh pose size is less than 40 .mu.m. The mesh size of the screens usually required to separate the various types of cells ranges from 20 .mu.m to 1 .mu.m. Sealed chambers occur when the liquid passing from the upper chamber, large mesh screen to a lower chamber wets the complete surface of the subjacent smaller mesh screen. Because of the high surface tension of the liquid between the pores of the mesh or screen, only the upper chambers fill completely leaving partially filled lower chambers. Liquid frequently "wicks" out of the device through the screens, creating large leaks and contamination of the contents of the unit. O-ring popping occurs because of unequal pressure on each side of the sleeve, causing leakage. Because the device must be disassembled from the top down, cells are exposed to air, which causes drying and contamination problems.
The object of the present invention is to solve the above-identified problems by providing a relatively simple cell separator for separating cells on the basis of size which is sealed and sterile during the separation process.